Method for preparing biological tissue

ABSTRACT

This invention provides a method for preparing biological tissue in which the thickness (i.e., the number of cell layers) can be easily regulated, culture can be conducted within a shorter period of time than is possible with conventional techniques, and addition of unfavorable components is not necessary. The method of the invention comprises adding a cell-containing culture solution to a culture vessel having a non-cell-adhesive inner bottom surface, conducting cell culture while centrifugal force toward the inner bottom surface is applied to the cells in the vessel, forming tissue via intercellular adhesion, and detaching and collecting the resulting tissue from the inner bottom surface.

TECHNICAL FIELD

The present invention relates to a method for preparing biologicaltissue that is suitable for preparation of biological tissue having amultilayer structure of a plurality of superposed cells in thethrough-thickness direction.

BACKGROUND ART

Biological tissue that is prepared via cell culture on a culture supportsuch as collagen has low cell density. Accordingly, such biologicaltissue is not suitable as transplant tissue for medical purposes. Atechnique for preparing tissue suitable for transplantation, such as acell sheet with high cell density, can be said to be an importanttechnique in the tissue engineering field. However, existing methods forpreparing biological tissue with high cell density have severaldrawbacks.

In general, sowing of cells on a culture support to form a cell sheet isextensively carried out. In order to facilitate detachment of a cellsheet, a technique of providing a layer of a temperature-responsivepolymer compound on a cell adhesion surface to accelerate celldetachment has been developed, and a cell sheet that is substantiallyfree from foreign matter can be collected. A cell sheet formed via suchtechnique, however, is generally composed of a single layer or three orfewer cell layers. Accordingly, it is necessary to superpose a pluralityof cell sheets in order to form a multilayer structure. Since a cellsheet is very thin and difficult to handle, superposition of a pluralityof such cell sheets is laborious rather than easy.

Patent Document 1 and Non-Patent Document 1 each disclose a method foreasily preparing a multilayered cell sheet in which cells are allowed tosupport magnetic fine particles thereon, magnetized cells are sowed in aculture vessel having a non-cell-adhesive bottom surface, the magnetizedcells are affixed to the bottom surface with a magnetic force, cellculture is conducted to form tissue, and tissue is collected via removalof the magnetic force at the end. This technique enables preparation ofmultilayered cells without the need for superposition of cell sheets.With this technique, however, contamination of biological tissue withforeign matter (i.e., magnetic fine particles) cannot be avoided. Thus,such technique cannot be said to be preferable for preparation oftransplant tissue for medical purposes.

Meanwhile, a method in which cells are exposed to centrifugal forceduring cell culture had been employed in the past for the purpose ofimposing stimuli to cultured cells. For example, Patent Document 2discloses a method for stimulating cells by regulating the dynamicenvironment with the application of hydrostatic pressure by centrifugalforce under cell culture conditions for the purpose of suppressing celldedifferentiation. Patent Document 2, however, does not refer to anymethod for detaching formed tissue. Also, what is disclosed in PatentDocument 2 is a method in which centrifugal force is intermittentlyapplied while growing cells over the period of several weeks. With suchmethod, a tissue mass of weakly adhering spherical cells, which arereferred to as spheroids, is formed (Patent Document 2, FIG. 6), and,thus, tissue that can be used as a graft, such as a cell sheet, cannotbe prepared.

Patent Document 3 discloses a method for preparing a three-dimensionalconstruct from bone cells or the like. According to the method of PatentDocument 3, a cell suspension is allowed to stand in a given cloningring, it is allowed to precipitate once, the cell suspension is allowedto stand further, tissue is formed from the precipitated cells, thetissue-forming cells are subjected to rotation culture so as to enhanceoxygen- and nutrition-diffusing effects, and a three-dimensionalconstruct of bone cells or the like with a centimeter-order size is thenprepared. However, such technique has drawbacks such that, for example,it takes several days to prepare tissue, target cells are limited tobone cells or the like, tissue cannot be easily detached from a culturevessel, and a conformation cannot be regulated as intended. Accordingly,biological tissue prepared by existing techniques disadvantageouslycontains unfavorable components, such as magnetic fine particles, andsuch biological tissue is not suitable for transplantation. Further, asufficient method for preparing biological tissue of superposed cellshas not yet been provided.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: WO 2004/083412

Patent Document 2: JP Patent Publication (kokai) No. 2004-81090 A

Patent Document 3: JP Patent No. 4084386

Non-Patent Document

Non-Patent Document 1: AKIRA ITO, et al., Tissue Engineering, Volume 10,Number 5/6, 873-880, 2004

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

It is an object of the present invention to provide a method forpreparing biological tissue in which a thickness (i.e., the number oflayers) can be easily regulated, culture can be conducted within ashorter period of time than existing techniques, and addition ofunfavorable components, such as magnetic fine particles, is notnecessary.

Means for Solving the Problem

Surprisingly, the present inventors found that such problem can besolved by adding a cell-containing culture solution to a culture vesselhaving an inner bottom surface, which is or can be converted into anon-cell-adhesive surface, and conducting culture by applyingcentrifugal force toward the inner bottom surface to the culturesolution. This has led to the completion of the present invention. Thepresent invention includes the following.

(1) A method for preparing tissue comprising:

a step of cell addition comprising adding a cell-containing culturesolution to a culture vessel having an inner bottom surface, which is orcan be converted into a non-cell-adhesive surface;

a step of cell culture comprising performing cell culture underconditions in which intercellular adhesion takes place while centrifugalforce toward the inner bottom surface is applied to the cells that wereadded to the culture vessel to form tissue via intercellular adhesion;and

a step of detachment comprising detaching and collecting the tissueobtained in the step of cell culture from the inner bottom surface.

(2) The method according to (1), wherein the number of cells in theculture solution is regulated in accordance with the number of celllayers in the through-thickness direction of tissue to be prepared.

(3) The method according to (1) or (2), wherein the culture solutioncontains an extracellular matrix component in a dissolved or dispersedstate.

(4) The method according to (3), wherein the extracellular matrixcomponent is prepared from cells derived from an individual identical tothat from which the cells to be cultured are derived.

(5) The method according to any of (1) to (4), wherein the inner bottomsurface of the culture vessel comprises a concave-convex patternprovided thereon.

(6) The method according to (5), wherein a configuration of theconcave-convex pattern enables detachment of tissue formed on the innerbottom surface of the culture vessel while maintaining the tissueconfiguration.

(7) The method according to (5) or (6), wherein the concave-convexpattern comprises a plurality of island-like convex portions andsea-like concave portions continuously formed around the convex portionsand the number of cells in a culture solution is regulated, so that thethickness of tissue to be prepared does not exceed the height of theconvex portion.

(8) The method according to any of (1) to (7), wherein all portions onthe inner surface of the culture vessel that are in touch with cells areor can be made non-cell-adhesive.

(9) Tissue prepared by the method according to any of (1) to (8).

(10) The tissue according to (9), wherein a plurality of cells aresuperposed in the through-thickness direction.

(11) The tissue according to (10), which comprises through-holes in thethrough-thickness direction.

Effects of the Invention

According to the method for preparing tissue of the present invention,the number of superposed cells in tissue can be easily regulated withoutthe addition of unfavorable components such as magnetic fine particles.

According to the method of the present invention, biological tissue canbe prepared within a shorter period of time compared with conventionaltechniques.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a procedure for preparing biological tissue according tothe present invention.

FIG. 2 shows a method for detaching and collecting a cell sheet formedon a non-cell-adhesive substrate.

FIG. 3 shows an extracellular matrix.

FIG. 4 shows an embodiment of preparation of biological tissue fortransplantation involving the use of cells and an extracellular matrixcollected from the same patient.

FIG. 5 shows a procedure for preparing biological tissue according to apreferable embodiment of the present invention.

FIG. 6 shows the non-cell-adhesive bottom surface of a culture vesselhaving a concave-convex pattern composed of island-like convex portionsand sea-like concave portions.

FIG. 7 schematically shows biological tissue prepared with the use ofthe culture vessel shown in FIG. 6 having the concave-convex pattern onthe bottom surface.

FIG. 8 shows a cell sheet prepared in Example 1.

FIG. 9 shows a cell sheet prepared in Example 1.

FIG. 10 shows that the cell sheet prepared in Example 1 has a multilayerstructure.

FIG. 11 shows the cell sheet prepared in Example 2.

FIG. 12 schematically shows a culture vessel having a concave-convexpattern on the inner bottom surface.

FIG. 13 shows a cell sheet prepared in Example 3 having through-holes.

FIG. 14 shows a cell sheet prepared in Example 4 having a concave-convexpattern on the surface.

FIG. 15 shows an embodiment of a concave portion with an inclined sidewall surface.

EMBODIMENTS FOR CARRYING OUT THE INVENTION 1. Step of Cell Addition

A step of cell addition involves addition of a cell-containing culturesolution to a culture vessel having inner surfaces, with at least theinner bottom surface being non-cell-adhesive or capable of being madenon-cell-adhesive.

A culture vessel used in the present invention may have inner surfacesof a vessel that accommodates a cell-containing culture solution, withat least the inner bottom surface being non-cell-adhesive or capable ofbeing made non-cell-adhesive. Particularly preferably, all surfaces thatare in contact with cells of the inner surfaces of a vessel thataccommodates a cell-containing culture solution (e.g., the inner bottomsurface and the inner side surface of the vessel) are or can be madenon-cell-adhesive.

Cells used in the present invention are not particularly limited,provided that such cells are cell adhesive. Examples of such cellsinclude: hepatic cells, which are hepatic parenchymal cells; endothelialcells, such as Kupffer cells, vascular endothelial cells, and cornealendothelial cells; epidermal cells, such as fibroblasts, osteoblasts,osteoclasts, periodontal ligament-derived cells, and epidermalkeratinocytes; epithelial cells, such as tracheal epithelial cells,alimentary epithelial cells, cervical epithelial cells, and cornealepithelial cells; muscle cells, such as alveolar epithelial cells,pericytes, smooth muscle cells, and cardiac muscle cells; nephrocytes;pancreatic Langerhans islet cells; nerve cells, such as peripheral nervecells, and optic nerve cells; cartilage cells; and bone cells. Thesecells may be primary cells that are directly collected from tissue ororgans, or they may be of cells lines established from such primarycells after several passages. Further, such cells may be any ofundifferentiated embryonic stem cells, pluripotent stem cells, such aspluripotent mesenchymal stem cells, unipotent stem cells, such asunipotent vascular endothelial progenitor cells, or differentiatedcells. Also, a single type of cells may be cultured or two or more typesof cells may be cultured together.

These cells are cultured via a conventional technique in advance, thecultured cells are treated with trypsin or the like, the resultants aresuspended in a culture solution, and the suspension is accommodated in aculture vessel in that state. Any culture solution can be used withoutparticular limitation, provided that it is a common medium used for cellculture in the art. In accordance with the type of a cell used, forexample, basal media described in “Soshiki Baiyou no Gijutsu” (TissueCulture Technique), vol. 3, p. 581, the Japanese Tissue CultureAssociation (ed.), Asakura Publishing Co., Ltd., such as MEM medium, BMEmedium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium,Fisher medium, F12 medium, WE medium, and RPMI1640 medium, can be used.Further, serum (e.g., fetal bovine serum), various growth factors,antibiotics, or amino acids may be added to the basal medium. Also,commercially available serum-free medium, such as Gibco serum-freemedium (Invitrogen), may be used. When clinical applications of cellulartissue that is obtained at the end are taken into consideration, use ofa medium free of animal-derived components is preferable.

In the present invention, an example of a non-cell-adhesive surface is ahydrophilic surface, and a specific example is a surface having a staticwater contact angle of 45 degrees or less at 20° C. Such surface can beobtained by forming a coating of an organic compound having acarbon-oxygen bond on the substrate surface. Alternatively, a substratemay be composed of a hydrophilic material.

A substrate material used for forming a hydrophilic coating on thesurface is not particularly limited. Specific examples include inorganicmaterials, such as metals, glass, ceramics, and silicon, and organicmaterials represented by elastomers and plastics (e.g., polyester resin,polyethylene resin, polypropylene resin, ABS resin, nylon, acrylicresin, fluorine resin, polycarbonate resin, polyurethane resin,methylpentene resin, phenol resin, melamine resin, epoxy resin, andvinyl chloride resin).

A non-cell-adhesive surface can be formed by a hydrophilic membranecomposed of an organic compound having a carbon-oxygen bond and having astatic water contact angle of 45 degrees or less.

The term “carbon-oxygen bond” refers to a bond formed between carbon andoxygen. Such bond may be a single or double bond. Examples ofcarbon-oxygen bonds include C—O bonds, C(═O)—O bonds, and C═O bonds.

Examples of the main raw materials for hydrophilic membranes includehydrophilic organic compounds, such as water-soluble polymers,water-soluble oligomers, water-soluble organic compounds, surface activematerials, and amphiphilic materials. When such materials are physicallyor chemically crosslinked to each other or such materials are physicallyor chemically bound to substrates, hydrophilic membranes are formed.

Specific examples of water-soluble polymer materials includepolyalkylene glycol and a derivative thereof, polyacrylic acid and aderivative thereof, polymethacrylic acid and a derivative thereof,polyacrylamide and a derivative thereof, polyvinyl alcohol and aderivative thereof, a zwitterionic polymer, and a polysaccharide.Examples of molecular shapes include linear polymers, branched polymers,and dendrimers. Specific examples thereof include, but are not limitedto, polyethylene glycol, a copolymer of polyethylene glycol andpolypropylene glycol (e.g., Pluronic F108, Pluronic F127),poly(N-isopropylacrylamide), poly(N-vinyl-2-pyrrolidone),poly(2-hydroxyethyl methacrylate), poly(methacryloyloxyethylphosphorylcholine), a copolymer of methacryloyloxyethylphosphorylcholine and an acrylic monomer, dextran, and heparin.

Specific examples of water-soluble oligomer materials and water-solublelow-molecular compounds include an alkylene glycol oligomer and aderivative thereof, an acrylic acid oligomer and a derivative thereof, amethacrylic acid oligomer and a derivative thereof, an acrylamideoligomer and a derivative thereof, a saponified product of a vinylacetate oligomer and a derivative thereof, an oligomer comprisingzwitterionic monomers and a derivative thereof, an acrylic acid and aderivative thereof, a methacrylic acid and a derivative thereof,acrylamide and a derivative thereof, a zwitterionic compound, awater-soluble silane coupling agent, and a water-soluble thiol compound.More specific examples include, but are not limited to, an ethyleneglycol oligomer, an (N-isopropyl acrylamide) oligomer, amethacryloyloxyethyl phosphorylcholine oligomer, low-molecular weightdextran, low-molecular weight heparin, oligoethylene glycol thiol,ethylene glycol, diethylene glycol, triethylene glycol, tetraethyleneglycol, 2-[methoxy(polyethyleneoxy)-propyl]trimethoxysilane, andtriethylene glycol-terminated-thiol.

The average thickness of a hydrophilic membrane is preferably 0.8 nm to500 μm, more preferably 0.8 nm to 100 μm, further preferably 1 nm to 10μm, and most preferably 1.5 nm to 1 μm. The average thickness of 0.8 nmor greater is preferable since the influence imposed by a region that isnot coated with the hydrophilic membrane on the substrate surface isinsignificant. When the average thickness is 500 μm or smaller, coatingis relatively easy.

Examples of methods for forming a hydrophilic membrane on a substratesurface include a method in which a hydrophilic organic compound isdirectly adsorbed to a substrate, a method in which a substrate isdirectly coated with a hydrophilic organic compound, a method in which asubstrate is coated with a hydrophilic organic compound followed bycrosslinking, a method in which a hydrophilic membrane is formed inmultiple steps so as to improve adhesion to a substrate, a method inwhich an underlying layer is formed on a substrate and the resultant isthen coated with a hydrophilic organic compound so as to improveadhesion to a substrate, and a method in which a polymerization originis formed on a substrate surface and a hydrophilic polymer brush is thenpolymerized.

Among such methods for forming membranes, particularly preferableexamples thereof include the method in which a hydrophilic membrane isformed in multiple steps and the method in which an underlying layer isformed on a substrate and the resultant is then coated with ahydrophilic organic compound so as to improve adhesion to a substrate.This is because adhesion of a hydrophilic organic compound to asubstrate can be easily improved via such techniques. In thisdescription, the term “bond layer” is used. The term “bond layer” refersto a layer that exists between the outermost hydrophilic membrane and asubstrate, when a coating of a hydrophilic organic compound is providedin multiple steps. When a hydrophilic membrane is provided on anunderlying layer provided on the substrate surface, the term refers tosuch underlying layer. A bond layer preferably contains a materialhaving a binding portion (i.e., a linker). Examples of combinations of alinker and a terminal functional group of a material to be bound to alinker include an epoxy group and a hydroxyl group, phthalic anhydrideand a hydroxyl group, a carboxyl group and N-hydroxysuccinimide, acarboxyl group and carbodiimide, and an amino group and glutaraldehyde.Either one in such combination may be a linker. In the above-describedmethods, a bond layer is formed on the substrate with the use of amaterial containing a linker prior to coating of the substrate with ahydrophilic material. The density of such material in the bond layer isan important factor that defines binding strength. Such density can beeasily evaluated by using a water contact angle on the bond layersurface as an indicator. In the case of a silane coupling agent havingan epoxy group at the terminus (i.e., epoxysilane), for example, whenthe substrate surface to which epoxysilane has been applied has a watercontact angle of typically 45 degrees or greater, and preferably 47degrees or greater, an ethylene glycol-based material or the like may beadded in the presence of an acid catalyst, so that a surface withsufficient non-cell-adhesive properties can be obtained.

Regions on the inner surfaces that are in contact with cells, includingthe inner bottom surface of the culture vessel used in the presentinvention, are preferably non-cell-adhesive, from the viewpoint of easeof detachment of tissue. A surface that is cell adhesive at the time ofcell culture but can be made non-cell-adhesive at the time of detachmentmay also be used. Such surface can be formed via immobilization of acovalent bond on a substrate surface by at least 1 type ofstimuli-responsive polymer selected from the group consisting of atemperature-responsive polymer, a pH-responsive polymer, and anion-responsive polymer. As a stimuli-responsive polymer, atemperature-responsive polymer is particularly preferable, although thepolymer is not limited thereto.

A temperature-responsive polymer that can be preferably used in thepresent invention exhibits hydrophobic properties at a temperature atwhich cells are cultured (about 37° C. in general) and exhibitshydrophilic properties at a temperature at which the cultured cell sheetis collected. A temperature at which a temperature-responsive polymer isconverted from hydrophobic into hydrophilic (i.e., a critical solutiontemperature T in water) is not particularly limited. From the viewpointof ease of collection of a cell sheet after culture, such temperature ispreferably lower than a temperature at which cells are cultured. Bycontaining such temperature-responsive polymer component, cellularscaffolds (i.e., cell-adhesion surfaces) are sufficiently retained atthe time of cell culture. Thus, cell culture can be efficiently carriedout. In contrast, a hydrophobic portion may be converted into ahydrophilic portion, and the cultured cell sheet is separated from acell culture substrate at the time of collection of a cell sheet afterculture, so that a cell sheet can be more easily collected. Atemperature-responsive polymer exhibiting hydrophilic properties at atemperature lower than a given critical solution temperature andexhibiting hydrophobic properties at a temperature higher than thecritical solution temperature is particularly preferable. The criticalsolution temperature of such temperature-responsive polymer isspecifically referred to as a lower limit critical solution temperature.

Specifically, a temperature-responsive polymer that can be preferablyused in the present invention has a lower limit critical solutiontemperature, T, of 0° C. to 80° C., and preferably 0° C. to 50° C. Tthat is higher than 80° C. is not preferable since cells may be killed.Also, T that is lower than 0° C. is not preferable since, in general,the cell growth rate is lowered to an extreme extent or cells may bekilled. Examples of such preferable polymers include acrylic andmethacrylic polymers. Specific examples of preferable polymers includepoly-N-isopropylacrylamide (T of 32° C.), poly-N-n-propylacrylamide (Tof 21° C.), poly-N-n-propyl methacrylamide (T of 32° C.),poly-N-ethoxyethylacrylamide (T of about 35° C.),poly-N-tetrahydrofurfuryl acrylamide (T of about 28° C.),poly-N-tetrahydrofurfuryl methacrylamide (T of about 35° C.), andpoly-N,N-diethylacrylamide (T of 32° C.). Examples of other polymersinclude poly-N-ethylacrylamide; poly-N-isopropyl methacrylamide;poly-N-cyclopropylacrylamide; poly-N-cyclopropyl methacrylamide;poly-N-acryloyl pyrrolidine; poly-N-acryloyl piperidine; polymethylvinyl ether; alkyl-substituted cellulose derivatives, such as methylcellulose, ethyl cellulose, and hydroxypropyl cellulose; polyalkyleneoxide block copolymers represented by a block copolymer ofpolypolypropylene oxide and polyethylene oxide; and polyalkylene oxideblock copolymers.

Examples of monomers used for forming such polymers include a monomerwhich can provide, when homopolymerized, T of 0° C. to 80° C. and can bepolymerized via radiation application. Examples of monomers include a(meth)acrylamide compound, an N-(or N,N-di)alkyl-substituted(meth)acrylamide derivative, a (meth)acrylamide derivative having acyclic group, and a vinyl ether derivative. At least one such monomermay be used. When a single type of monomer is used alone, a polymerformed on the substrate is a homopolymer. When a plurality of types ofmonomers are used, a polymer formed on the substrate is a copolymer.Both types of polymers are within the scope of the present invention.When regulation of T is necessary in accordance with the type of growncell, improvement in the interaction between a coating material and acell culture support is necessary, or adjustment of thehydrophilic/hydrophobic balance of a cell support is necessary, forexample, monomers other than the above monomers may further be added,and copolymerization may be carried out. Further, a graft or blockcopolymer of the above-described polymer used in the present inventionand another polymer or a mixture of the polymer of the present inventionand another polymer may be used. Also, crosslinking may be carried outwhile maintaining properties inherent to the polymer.

A pH-responsive polymer and an ion-responsive polymer that are suitablefor preparation of a cell sheet can be adequately selected.

2. Step of Cell Culture

The step of cell culture comprises performing cell culture underconditions in which intercellular adhesion takes place while centrifugalforce toward the inner bottom surface is applied to the cell-containingculture solution accommodated in a culture vessel and forming tissue viaintercellular adhesion. FIGS. 1A and 1B each represent this step. Inthis step, cells are in close contact with the inner bottom surface withthe aid of centrifugal force in accordance with the configuration of theinner bottom surface, and cells adhere to each other in such state.Thus, tissue of a desired configuration is formed.

The magnitude of centrifugal force can be adequately selected within arange such that tissue can be formed without adversely affectingcellular functions. For example, centrifugal force is preferably 2 G to1440 G, and more preferably 2 G to 720 G. By mounting a culture vesselthat accommodates a cell-containing culture solution in a centrifuge andperforming centrifugation, centrifugal force can be applied.

The term “conditions in which intercellular adhesion takes place” refersto conditions in which cells act and cells can adhere to each other. Forexample, temperature is preferably 20° C. to 40° C., atmospheric gasconcentration is preferably 3% to 5% carbon dioxide, and cultureduration is preferably 0.5 to 24 hours, although such conditions vary inaccordance with the type of a cell to be cultured. An advantage of thepresent invention is that culture duration can be shortened and a damageimposed on cells can be reduced. Culture duration can be shortenedaccording to an embodiment described below in which an extracellularmatrix is separately prepared and added, and culture duration can beshortened to 0.5 to 3 hours. Thus, such embodiment is preferable.According to an embodiment in which an extracellular matrix is notadded, culture duration can be 1 to 24 hours.

In the examples, a culture vessel was allowed to stand for severalminutes in an incubator filled with an atmospheric gas of interest (5%carbon dioxide), the culture vessel was covered with a lid so as tomaintain the atmospheric gas conditions, and culture under additionalgravity was then conducted. However, culture is not limited to theabove-described methods. For example, culture under additional gravitycan be carried out while maintaining the culture vessel in an open statein an incubator in which the atmospheric gas and temperature conditionsare regulated.

In the step of cell culture, it is sufficient if intercellular adhesiontakes place, and an increase in the number of cells via multiplicationis not essential. This is because the thickness of tissue prepared(i.e., the number of cell layers in the through-thickness direction oftissue) can be controlled by adequately regulating the number of cellsin a culture solution. Since cell multiplication is not necessary in thestep of cell culture, tissue can be obtained within a relatively shortperiod of time. Also, tissue thickness and configuration can be freelyregulated.

According to the method of the present invention, tissue comprisingdensified cells can be obtained. Such tissue comprising densified cellsis preferable for transplantation.

3. Step of Detachment

The step of detachment comprises detachment and collection of tissuesobtained after centrifugation from the inner bottom surface of theculture vessel. FIG. 1C represents this step. For example, cells can bedetached via a physical operation such as pipetting as described in theexamples. If the inner bottom surface of the culture vessel isnon-cell-adhesive, such operation is easy, and tissue can be prepared,detached, and collected via culture in a short period of time. When theinner bottom surface of the culture vessel is a surface which can beconverted into a non-cell-adhesive surface, such as a stimuli-responsivepolymer, a detachment operation is carried out in the environment inwhich a surface can be converted into a non-cell-adhesive surface (e.g.,at or below the lower limit of the critical temperature).

Detachment may be carried out via a known method as shown in FIG. 2while maintaining tissue configuration. In FIG. 2, gelatin is firstintroduced onto the tissue surface formed on a non-cell-adhesivesubstrate for gelation, gelatin gel is held by a holding substrate, suchas a PET film (FIG. 2B), and tissue is then detached and collected fromthe non-cell-adhesive substrate with gelatin gel (FIG. 2C). Thethus-detached and collected tissue is brought into close contact withthe target of transplantation (FIG. 2D), gelatin is dissolved at 37° C.(FIG. 2E), and the tissue can then be transplanted to the target ofinterest.

4. More Preferable Embodiment 1 of the Present Invention

According to the step of cell culture of the present invention, cellsare brought into close contact to each other with the aid of centrifugalforce, and cells adhere to each other via an extracellular matrixsecreted from the cells within several hours (FIG. 3). In general, ittakes several hours for the cultured cells to secrete the extracellularmatrix.

According to a more preferable embodiment of the present invention, asolution or dispersion of an extracellular matrix component that wasseparately prepared is added to a culture solution comprising cellssuspended therein. Specifically, a culture solution preferably comprisesan extracellular matrix component dissolved or dispersed therein.Addition of an extracellular matrix component that was separatelyprepared can accelerate the speed of intercellular adhesion. Accordingto this embodiment, a step of cell culture can be carried out within 0.5to 3 hours, tissue preparation can be carried out more efficiently, andthe damage imposed on tissue can be minimized.

An extracellular matrix is prepared from a cell. A cell used forpreparing an extracellular matrix is preferably derived from anindividual identical to that from which the cells to be cultured arederived. Since all the biological components of the tissue that isprepared at the end are derived from the same individual, tissuetransplantation can be highly safe. FIG. 4 shows a process of preparingtissue by the method of the present invention and transplanting theprepared tissue into a patient. As shown in FIG. 4, it is preferablethat cells are collected from a single patient, the collected cells arecultured, a cell suspension is prepared from a culture product, anextracellular matrix-containing solution is prepared from anotherculture product, both the cell suspension and the solution are used tocarry out the aforementioned steps of cell addition, cell culture, anddetachment to prepare tissue, and the obtained tissue is thentransplanted into the patient.

Cells used for preparing an extracellular matrix are not particularlylimited, provided that such cells can form an extracellular matrix.Examples of such cells include adhesive cells, and specific examplesinclude cells selected from the same group as the group exemplified inthe present description as specific examples of cells to be cultured.Cells used for preparing an extracellular matrix are not necessary thesame as those to be cultured, although cells of the same type arepreferable.

An extracellular matrix component is not necessarily used with anextracellular matrix being purified. Tissue comprising many cellsadhered thereto with the aid of an extracellular matrix (e.g., a cellsheet formed on a cell-adhesive substrate) is ground, the ground productis fragmented via ultrasound application, solid matter is separated viacentrifugation or via other means, according to need, and the resultingextracellular-matrix-containing solution can be used as an extracellularmatrix component. The extracellular-matrix-containing solution comprisesan extracellular matrix component dissolved or dispersed therein.

5. More Preferable Embodiment 2 of the Present Invention

In the present invention, further, a concave-convex pattern ispreferably formed on the inner bottom surface of the culture vessel.According to this embodiment, biological tissue having a desiredthree-dimensional configuration is prepared using a concave-convexpattern as a template (FIG. 5A to 5C).

Preferably, a concave-convex pattern enables detachment of tissue formedon the inner bottom surface of the culture vessel (such tissue isprepared using a concave-convex pattern as a template and thus has atleast in part an inverted pattern of the concave-convex pattern) whilemaintaining the tissue configuration. Typical examples of suchconfigurations include a concave-convex pattern in which the side wallsof the concave portion on the cross-section of the concave portion alongwith a plane vertical to the bottom surface of the culture vessel areprovided parallel to the axis passing through the space inside theconcave portion and being vertical to the bottom surface of the culturevessel (e.g., the configuration shown in FIG. 5), a concave-convexpattern in which a distance between such axis and the side wall of theconcave portion becomes larger from a closed end of the concave portiontoward an open end thereof (e.g., the configuration shown in FIG. 15),and a concave-convex pattern comprising both such configurations. Suchconfiguration of the concave portion is preferable since suchconfiguration enables detachment of the formed tissue from theconcave-convex pattern without destruction of the inverted pattern oftissue.

A particularly preferable concave-convex pattern comprises a pluralityof island-like convex portions and sea-like concave portionscontinuously formed around such convex portions. An example of suchconcave-convex pattern is shown in FIG. 6. With the use of the culturevessel shown in FIG. 6 having the concave-convex pattern at the bottom,cells are accumulated in the concave portion due to centrifugal force,and such cells adhere to each other. When the number of cells in theculture solution is regulated so as to prevent the thickness of theprepared tissue from exceeding the height of the convex portion,lattice-like tissue having through-holes in the through-thicknessdirection is formed as shown in FIG. 7. Such through-holes function asroutes of nutrition supply to the inside of tissue and as routes ofwaste discharge. If cell sheets without through-holes are merelysuperposed, cellular necrosis takes places. If through-holes are formedaccording to the present embodiment, however, cellular necrosis can besuppressed. While through-holes could be formed on a monolayer cellsheet according to conventional techniques, through-holes had to bealigned with each other in order to form a multilayer structure. Sincethrough-holes are very fine, there is no practical means for realizingalignment thereof at present. The present embodiment is very usefulsince formation of multilayered cells and formation of a fluid passagecan be simultaneously carried out.

As shown in FIG. 14 (Example 4), the number of cells in the culturesolution may be regulated so as to cover the top surfaces of concaveportions as well as convex portions on the inner bottom surface of theculture vessel with cells, upon application of centrifugal force. Thus,an inverted pattern of the concave-convex pattern on the inner bottomsurface of the culture vessel can be formed on the tissue surface.

Another embodiment of a concave-convex pattern is a concave-convexpattern comprising convex ribs and concave grooves that are alternatelyarranged in parallel.

A method for forming a concave-convex pattern is not particularlylimited, and a concave-convex pattern can be formed via general fineprocessing technologies.

Example 1 1. Preparation of Cell Sheet with Centrifugal Force

1-1. Preparation and Collection of Cell Sheet with Centrifugal Force

Polyethylene glycol was chemically applied to the entire inner surfaceof a glass vessel having a bottom with a diameter of 2 cm to render theentire inner surface non-cell-adhesive, 2 ml of a cell suspensioncontaining 4×10⁶ mouse fibroblasts suspended in 10% fetal bovineserum-containing DMEM medium was sowed in the vessel, and culture wasconducted at 37° C. in the presence of 5% CO₂ for 3 hours while applyingcentrifugal force of 720 G toward the bottom (FIG. 8A). After culture,centrifugation was terminated, and a cell sheet was easily and rapidlydetached and collected from the substrate without destruction of thetissue structure by a physical force, such as via pipetting (FIG. 8B).As a contrast, culture was conducted in the same manner, except that nocentrifugal force was applied. As a result, a cell sheet could not beprepared, cells were detached from each other via pipetting, and thetissue structure was destroyed.

1-2. Observation and Live/Dead Assay of Three-Dimensional Tissue

The cell sheet collected in 1-1 above was transferred to a culture dishfor observation and observed. As a result, a cell sheet having adiameter of 2 cm and the same configuration as that of the vessel wasobtained (FIG. 9). Also, cells were stained with calcein and subjectedto cross-sectional observation under a confocal microscope. As a result,a thickness of the cell sheet was found to be about 3 or 4 cell layers(FIG. 10). In order to inspect the cell survival of the obtained cellsheet, the cell sheet was treated with trypsin and EDTA to dispersecells, and cell viability was assayed using a cell live/dead assay kit(product name: Cell double staining kit; manufacturer: DojindoLaboratories; product number: CS01). As a result, the cell viability was90% before application of centrifugal force in 1-1, and the cellviability of the resulting cell sheet was also 90%. The results verifythat cells would not be substantially killed by the operation of 1-1.

Example 2 2. Addition of Extracellular Matrix

2-1. Preparation of Extracellular Matrix-Containing Solution

DMEM medium containing 10% fetal bovine serum was added to a 10-cmpolystyrene petri dish to culture mouse fibroblasts, and the cells weregrown to reach confluence in order to have the cells to produce anextracellular matrix. Subsequently, multiplied cells were ground using acell scraper, the cells were completely fragmented via ultrasoundtreatment, the fragmented product was centrifuged, and the supernatantwas collected. The supernatant was used below as an extracellularmatrix-containing solution.

2-2. Preparation and Collection of Cell Sheet with Centrifugal Force

Polyethylene glycol was chemically applied to the entire inner surfaceof a glass vessel having a bottom with a diameter of 2 cm to render theentire inner surface non-cell-adhesive, 2 ml of a cell suspensioncontaining 4×10⁶ mouse fibroblasts suspended in the extracellularmatrix-containing solution prepared in 2-1 was sowed in the vessel, andculture was conducted at 37° C. in the presence of 5% CO₂ for 1 hourwhile applying centrifugal force of 720 G toward the bottom (FIG. 11A).After culture, centrifugation was terminated, and a cell sheet waseasily and rapidly detached and collected from the substrate withoutdestruction of the tissue structure by a physical force, such as viapipetting (FIG. 11B). As a contrast, culture was conducted in the samemanner, except that a DMEM medium containing 10% fetal bovine serum wasused instead of the extracellular matrix-containing solution prepared in2-1. As a result, a cell sheet could not be prepared, cells weredetached from each other via pipetting, and the tissue structure wasdestroyed (e.g., the cell sheet was torn).

Example 3 3. Preparation of Cell Sheet Having Holes

3-1. Culture Vessel Having Non-Cell-Adhesive Bottom Surface withConcave-Convex Configuration

A silicon wafer was coated with resist (tradename: SU-8; manufacturer:MicroChem) to a thickness of 200 μl, a concave-convex template wasprepared via optical lithography, a 3% agarose solution was introducedonto the template for gelation, and the resulting gel was detached fromthe template. Thus, agarose gel having a concave-convex pattern (200μm×200 μm squares raised at heights of 200 μm at intervals of 200 μm)was prepared. The resulting gel was positioned at the bottom of thevessel, with the entire inner bottom surface being madenon-cell-adhesive in 1-1, so that the concave-convex surface of the gelwould face upward. Thus, a culture vessel having a non-cell-adhesive andconcave-convex bottom surface was prepared (FIG. 12).

3-2. Preparation of Extracellular Matrix-Containing Solution

DMEM medium containing 10% fetal bovine serum was added to a 10-cm petridish to culture mouse fibroblasts, and the cells were grown to reachconfluence in order to have the cells to produce an extracellularmatrix. Subsequently, cells were ground using a cell scraper, the cellswere completely fragmented via ultrasound treatment, the fragmentedproduct was centrifuged, and the supernatant was collected. Thesupernatant was used below as an extracellular matrix-containingsolution.

3-3. Preparation and Collection of Cell Sheet with Centrifugal Force

A cell suspension (2 ml) containing 10×10⁶ mouse fibroblasts suspendedin the extracellular matrix-containing solution prepared in 3-2 wassowed in the vessel prepared in 3-1, and culture was conducted at 37° C.in the presence of 5% CO₂ for 3 hours while applying centrifugal forceof 720 G toward the bottom. Thereafter, centrifugation was terminated,and cell sheets having holes were easily and rapidly detached andcollected from the substrate while maintaining the configuration andrefraining from destruction of the tissue structure by a physical force,such as via pipetting (FIG. 13).

3-4. Live/Dead Assay of Cell Sheet Having Holes

The cell sheet having 200 μm×200 μm square-shaped holes at intervals of200 μm prepared in 3-3 was transferred to a polystyrene dish for cellculture, the cell sheet was cultured for 4 days, the cell sheet wastreated with trypsin and EDTA to disperse cells, and cell viability wasassayed using a cell live/dead assay kit (product name: Cell doublestaining kit; manufacturer: Dojindo Laboratories; product number: CS01).The cell viability was 86%. Also, a cell sheet without holes wasprepared with the use of a culture vessel having the same type of flatbottom surface as that of 3-3, the cell sheet was also cultured for 4days, and the cell viability was assayed and found to be 70%.

Example 4 Preparation of Cell Sheet Having Patterned Configuration

In the same manner as in 3-1 above, a vessel having a bottom surface(such bottom surface comprising a plurality of parallel lines providedat intervals of 300 μm, raised at heights of 50 μm, and having widths of200 μm) was prepared using a concave-convex template coated with resist(thickness: 50 μm). The thus-prepared vessel was used to prepare a cellsheet in the same manner as in 3-3, except that cell density wasregulated so as to cover the convex ribs as well as lined concavegrooves with cells. Thus, a cell sheet having a patterned configurationwas prepared (FIG. 14).

The invention claimed is:
 1. A method for preparing a tissue comprising:providing a cell suspension in a culture vessel with a non-cell adhesivebottom, wherein said cell suspension comprises animal cells suspended ina culture medium; applying a centrifugal force that is directed towardthe inner bottom surface of the culture vessel, having a predefinedshape, wherein the culture medium is retained in the culture vesselduring centrifugation and said centrifugal force deposits a plurality ofcells on the inner bottom surface; culturing said cells deposited on theinner bottom surface in the presence of said centrifugal force for aperiod of time sufficient to allow neighboring cells to adhere to eachother, wherein said culturing in the presence of said centrifugal forceproduces a tissue comprising a plurality of continuous cell sheets,having a shape that is defined by the predefined shape of the innerbottom surface of the culture vessel.
 2. The method for preparing atissue according to claim 1, wherein the thickness of the tissue on theinner bottom surface of the culture vessel is determined by the numberof cells in the cell suspension.
 3. The method for preparing a tissueaccording to claim 1, wherein the culture medium comprises isolatednatural extracellular matrix in a dissolved or dispersed state tofacilitate intercellular adhesion.
 4. The method for preparing a tissueaccording to claim 3, wherein the isolated, natural extracellular matrixand the cells in the cell suspension are isolated from a tissue takenfrom the same individual.
 5. The method for preparing a tissue accordingto claim 1, wherein the inner bottom surface of the culture vesselcomprises a concave-convex pattern.
 6. The method for preparing a tissueaccording to claim 5, wherein said concave-convex pattern permitsdetachment of the tissue formed on the inner bottom surface of theculture vessel while preserving the tissue's shape.
 7. The method forpreparing a tissue according to claim 5, wherein the concave-convexpattern comprises a plurality of island-like convex portions and aplurality of sea-like concave portions, wherein the concave portions aredistributed continuously around the convex portions, and wherein thenumber of cells in the cell suspension forms a tissue with a thicknessthat does not exceed the height of any island-like convex portion. 8.The method for preparing a tissue according to claim 1, wherein thesteps of claim 1 are repeated with a cell suspension having cells of adifferent cell type.
 9. The method for preparing a tissue according toclaim 1, wherein the cells are cultured in the presence of saidcentrifugal force for between about 0.5 hours and about 3 hours.